Lsm 5 image examiner software

Post hoc image analysis was performed using LSM 5 Image Examiner software (Zeiss) and ImageJ software (NIH) in a blinded fashion to evaluate the changes in image intensity, astrocyte number, and morphological features of astrocytes over time. A standard area of 150 μm×150 μm was chosen as the region of interested (ROI) for each mouse, and the same ROI was analyzed at different time points. Image intensity was measured as mean GFP fluorescence intensity under the same excitation laser power and acquisition settings, with follow-up images over time normalized to the baseline image. Astrocyte number was counted in the same ROI at different time points. Morphological features of astrocytes were assessed with respect to total astrocyte size (including processes) and soma size, based on area calculations from the projected Z-stacks (S1 Fig).

Sections were treated with 3% H₂O₂ for 10 min at room temperature to quench endogenous peroxidase activity. Antigen retrival was achieved by heating sections immersed in sodium citrate buffer (pH 6.0) for 10 min using a microwave oven. The sections were incubated overnight (4°C) with primary antibodies against GFAP at 1:800 and ICAM-1 at 1:100. The sections were washed and then incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (1:300, Santa Cruz). Color reaction was achieved by DAB incubation for 3 min at room temperature. The sections were then rinsed in distilled water and counterstained with hematoxylin. The sections were cover-slipped and analyzed using a computer image analysis system (Zeiss LSM5 Image Examiner software).
The immunofluorescent double labeling of ICAM-1 with the astrocyte marker GFAP or with the neuronal marker NeuN was also performed using the afore-mentioned antibodies. The double labeling of ICAM-1 and GFAP or ICAM-1 and NeuN was achieved by incubating with the two primary antibodies separately and then with a mixture of the two fluorescence-conjugated secondary antibodies. The specimens were mounted with Vectashield Hardset Mounting Media (H-1200, Vector) containing 4′, 6-diamidino-2-phenylindole (DAPI) and examined using a fluorescence confocal-scanning microscope (Nikon Eclipse C1).

Immunofluorescence and morphometric analyses were executed as described below. SH-SY5Y cells were plated with a density of 75 × 10³/well in a 24 wells plate, grown on a glass coverslip (coated with poly-l-lysine, Sigma-Aldrich); IMR-32 cells were plated with a density of 50 × 10³/well and grown on a glass coverslip coated with collagen IV (BD Bioscience). Cells were fixed in ice-cold methanol (Sigma-Aldrich), then washed and incubated in Phosphate Buffered Saline (PBS, Sigma-Aldrich) containing 1% of Bovine Serum Albumin (BSA, Sigma-Aldrich) and 0.2% Triton X 100 overnight at 4 °C with a polyclonal anti-β III tubulin (Sigma-Aldrich, 1:600), monoclonal anti-Notch 1 (Sigma-Aldrich, 1:1000). After rinses, cells were incubated with Alexa Fluor® 488 (Life Technologies, 1:400) anti rabbit secondary antibody and CY™3-conjugated anti-mouse secondary antibody (Jackson Immunoresearch Laboratories INC.,1:500) in PBS for 1 h at room temperature. For morphological evaluation, slices were mounted and examined by a ZEISS LSM 510 META confocal laser-scanning microscope (Carl Zeiss, Germany). Images were processed using LSM5 image examiner software (Zeiss). The percentage of morphologically differentiated cells was determined by analyzing at least 10 fields for each treatment; cells with neurites ≥50 μm in length were considered as differentiated.